ACBD4 Antibody

What is ACBD4 and why is it significant in cellular research?

ACBD4 is a member of the acyl-CoA binding domain (ACBD) family, characterized by the presence of an N-terminal acyl-CoA binding domain. This protein has gained significance in cellular research as it functions as a molecular tether between peroxisomes and the endoplasmic reticulum (ER). Specifically, ACBD4 isoform 2 has been identified as a tail-anchored peroxisomal membrane protein that interacts with the ER-resident protein VAPB to facilitate interaction between these two organelles .

The discovery of ACBD4 as a peroxisome-ER tether is particularly significant because it represents only the second protein identified to be involved in peroxisome-ER contacts in mammals, alongside ACBD5 . This finding enhances our understanding of organelle communication and the functional significance of membrane contact sites in cellular physiology.

How does ACBD4 differ from other members of the ACBD family, particularly ACBD5?

While ACBD4 and ACBD5 share 58% sequence identity, this similarity is primarily restricted to their N-terminal acyl-CoA binding domains, with the remainder of the proteins exhibiting significant differences . The key differences include:

These structural differences likely translate to functional distinctions, with ACBD5 demonstrating more efficient FFAT-mediated interactions with VAP proteins compared to ACBD4 .

What are the known isoforms of ACBD4 and how can I detect them with antibodies?

ACBD4 has three major isoforms as defined by UniProt (identifier: Q8NC06) . The most well-characterized is isoform 2 (UniProt identifier: Q8NC06-2), which contains:

• An N-terminal acyl-CoA binding domain

• A potential coiled-coil domain

• A predicted FFAT-like motif

• A C-terminal transmembrane domain (TMD) and tail

When selecting antibodies for ACBD4 detection, researchers should consider:

• Epitope location relative to isoform-specific regions

• Cross-reactivity potential between isoforms

• Validation in tissues known to express ACBD4 positively and negatively

• Application-specific optimization for Western Blot, Immunohistochemistry, or Immunocytochemistry/Immunofluorescence

Commercial antibodies are available that have been validated for various research applications and are reactive in human samples .

Where does ACBD4 localize subcellularly and how can this be visualized?

• At normal expression levels: Exclusively peroxisomal localization

• At high expression levels: Peroxisomal localization with altered peroxisome morphology and additional weak mitochondrial signal

This dual localization phenomenon has been observed for other peroxisomal tail-anchored proteins such as Pex26 . To visualize ACBD4 localization, researchers typically use:

• Fluorescently-tagged ACBD4 constructs (GFP-ACBD4iso2, Myc-ACBD4iso2, FLAG-ACBD4iso2)

• Co-localization with organelle markers:

  • Peroxisomes: PEX14, catalase

  • Mitochondria: Tom20

Confirmation of ACBD4's topology as a tail-anchored protein with the N-terminus exposed to the cytosol can be achieved through differential permeabilization experiments using digitonin (which preserves peroxisomal membranes) and Triton X-100 (which permeabilizes all membranes) .

How can I investigate ACBD4-mediated protein interactions using antibody-based approaches?

Investigating ACBD4 protein interactions requires several complementary approaches:

Immunoprecipitation strategy:

• Express tagged versions of ACBD4 (e.g., GFP-ACBD4iso2) and potential interacting partners (e.g., Myc-VAPB) in appropriate cell lines

• Perform pull-down using tag-specific magnetic beads (e.g., GFP-Trap or Myc-TRAP)

• Analyze precipitated proteins by immunoblotting with appropriate antibodies

For identification of novel interacting partners, mass spectrometry analysis of immunoprecipitated complexes has proven effective. This approach identified VAPA and VAPB as ACBD4-interacting proteins in a proteomics study using GFP-ACBD4iso2 pull-downs .

Essential controls include:

• Negative control: GFP alone for GFP-tagged constructs

• Positive control: Known interaction partners (e.g., GFP-ACBD5 for VAPB interaction studies)

What are the optimal fixation and permeabilization methods for ACBD4 immunostaining?

Successful immunostaining of ACBD4 depends on preserving both its structure and interactions while enabling antibody access. Based on published protocols:

Differential permeabilization is particularly useful for topology studies. For example, the N-terminal FLAG-tag of FLAG-ACBD4iso2 was detectable after digitonin treatment, confirming its cytosolic orientation .

How can I design experiments to distinguish the functions of ACBD4 from ACBD5?

Despite their similarities, ACBD4 and ACBD5 likely have distinct functions. To distinguish between them:

• Comparative binding assays: Quantify relative binding affinities to VAPB using techniques like:

  • Surface plasmon resonance

  • Microscale thermophoresis

  • Fluorescence polarization

• FFAT motif analysis: Create targeted mutations in the FFAT motifs of both proteins to assess:

  • The role of the acidic tract (SDSDS in ACBD5 vs. modified sequence in ACBD4)

  • Contribution of specific residues to binding efficiency

• Domain swapping experiments: Generate chimeric proteins to identify functional domains:

  • ACBD4 with ACBD5 FFAT motif

  • ACBD5 with ACBD4 FFAT motif

• Localization studies under variable expression conditions: Compare how expression levels affect:

  • Peroxisome morphology

  • Mitochondrial mistargeting

  • ER-peroxisome contact sites

How can I validate the specificity of ACBD4 antibodies?

Antibody specificity is crucial for reliable results. A comprehensive validation strategy includes:

• Positive and negative tissue controls: Test antibodies on tissues known to express ACBD4 positively and negatively

• Knockdown/knockout validation:

  • siRNA/shRNA knockdown of ACBD4

  • CRISPR/Cas9 knockout cell lines

  • Compare antibody signal between control and ACBD4-depleted samples

• Recombinant protein controls:

  • Pre-absorption with recombinant ACBD4

  • Blocking peptide controls

• Multiple antibody approach: Use antibodies targeting different epitopes to confirm results

• Isoform specificity testing: Validate against samples expressing different ACBD4 isoforms

What controls are essential when performing co-localization studies with ACBD4 antibodies?

For reliable co-localization studies involving ACBD4, include these essential controls:

• Single channel controls: Image each fluorophore separately to assess bleed-through

• Organelle markers:

  • Peroxisomes: PEX14, catalase

  • ER: VAPB, PDI, calnexin

  • Mitochondria: Tom20

  • These markers help distinguish true co-localization from coincidental overlap

• Expression level controls:

  • Low, medium, and high expression samples

  • Important since ACBD4 localization changes with expression level

• Quantitative analysis:

  • Pearson's correlation coefficient

  • Manders' overlap coefficient

  • Line scan analysis across organelles

• Resolution controls: Include samples with known degrees of co-localization to calibrate analysis methods

How can I investigate the role of ACBD4 in peroxisome-ER tethering?

Investigating ACBD4's tethering function requires multiple complementary approaches:

• Microscopy-based methods:

  • Confocal microscopy of cells co-expressing ACBD4 and VAPB shows characteristic peroxisome-ER associations

  • Super-resolution microscopy for detailed contact site analysis

  • Live-cell imaging to monitor dynamics of tethering

• Biochemical approaches:

  • Proximity labeling (BioID, APEX) to identify proteins at contact sites

  • Subcellular fractionation to isolate contact site-enriched fractions

  • Cross-linking mass spectrometry to capture transient interactions

• Functional assays:

  • Lipid transfer assays between peroxisomes and ER

  • Calcium signaling at contact sites

  • Peroxisomal metabolism assessments

• Perturbation experiments:

  • FFAT motif mutations to disrupt VAPB binding

  • TMD mutations to alter peroxisomal targeting

  • Overexpression of competing fragments

When ACBD4 and VAPB are co-expressed, researchers observed increased ER-peroxisome associations that allowed visualization of discrete peroxisomal structures using VAPB as an ER marker . Interestingly, when ACBD4 mistargeted to mitochondria, researchers detected increased association of VAPB-labeled ER with mitochondria, suggesting ACBD4 can mediate ER-mitochondria interactions when mislocalized .

What approaches can reveal the dynamics of ACBD4 localization under different cellular conditions?

To study dynamic aspects of ACBD4 localization:

• Live-cell imaging techniques:

  • FRAP (Fluorescence Recovery After Photobleaching) to measure mobility

  • Photo-switchable fluorescent tags to track movement between compartments

  • Dual-color imaging with organelle markers

• Inducible expression systems:

  • Tet-On/Off for controlled expression levels

  • Optogenetic control of ACBD4 expression

  • Degradation domain fusion for rapid protein depletion

• Stress response experiments:

  • Oxidative stress (H₂O₂, paraquat)

  • ER stress (tunicamycin, thapsigargin)

  • Nutrient deprivation

  • Monitor changes in ACBD4 localization and peroxisome-ER contacts

• Cell cycle analysis:

  • Synchronization methods

  • Cell cycle markers

  • Time-lapse imaging through division

What are the key considerations when studying ACBD4 in different model systems?

Different experimental models present unique challenges for ACBD4 research:

Species-specific considerations are important as mutations in the N-terminal region of the ACBD4 FFAT motif have been observed between mammals and birds, altering the polarity of the acidic tract .

How can ACBD4 antibodies be used to investigate potential disease mechanisms?

While direct links between ACBD4 and specific diseases are not established in the provided search results, several antibody-based approaches can explore potential disease connections:

• Expression analysis in disease states:

  • Immunohistochemistry of patient samples

  • Western blot quantification in disease models

  • Single-cell analysis of expression heterogeneity

• Post-translational modification studies:

  • Phospho-specific antibodies

  • Ubiquitination detection

  • Other PTM-specific antibodies to detect disease-associated modifications

• Proximity studies with disease-associated proteins:

  • Proximity ligation assay (PLA)

  • FRET/FLIM analysis

  • Co-immunoprecipitation under disease-mimicking conditions

• Organelle pathology assessment:

  • Peroxisome morphology in disease states

  • ER-peroxisome contact site quantification

  • Correlative light and electron microscopy with immunogold labeling

Of particular interest would be investigating ACBD4 in disorders affecting peroxisome function or ER-peroxisome communication, given its established role in tethering these organelles.